COVID-19: A unique feature has been discovered that could explain why it is so transmissible among people

Cornell University researchers studying the structure of the virus that causes COVID-19 have found a unique feature that could explain why it is so transmissible between people.

Researchers also note that, aside from primates, cats, ferrets and mink are the animal species apparently most susceptible to the human virus.

Gary Whittaker, professor of virology, is the senior author on the study, which identifies a structural loop in the SARS-CoV-2 spike protein, the area of the virus that facilitates entry into a cell, and a sequence of four amino acids in this loop that is different from other known human coronaviruses in this viral lineage.

An analysis of the lineage of SARS-CoV-2 showed it shared properties of the closely related SARS-CoV-1, which first appeared in humans in 2003 and is lethal but not highly contagious, and HCoV-HKU1, a highly transmissible but relatively benign human coronavirus. SARS-CoV-2 is both highly transmissible and lethal.

“It’s got this strange combination of both properties,” Whittaker said. “The prediction is that the loop is very important to transmissibility or stability, or both.”

Whittaker said the researchers are focused on further study of this structural loop and the sequence of four amino acids.

Cats, ferrets and minks are also susceptible. In order to infect a cell, features of the spike protein must bind with a receptor on the host cell’s surface, and cats have a receptor binding site that closely matches that of humans.

To date, infections in cats appear to be mild and infrequent, and there is not evidence that cats can, in turn, infect humans.

Whittaker added that investigations into feline coronaviruses could provide further clues into SARS-CoV-2 and coronaviruses in general.

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In this study, we perform phylogenetic, bioinformatic, and homology structural modeling analyses of SARS-CoV-2 S, in comparison with closely related viruses. We identify a distinct four residue insert (featuring two arginine residues) that maps to the S1/S2 priming loop of SARS-CoV-2, which is missing from all other SARS-CoV-related viruses but present in MERS-CoV S and in many other coronaviruses.

We discuss the importance of this extended basic loop for S protein-mediated membrane fusion and its implications for virus transmission.

ResultsComparison of amino acid identity of the spike (S) protein of SARS-CoV-2 with human SARS-CoV

To obtain an initial assessment of shared and/or specific features of the SARS-CoV-2 spike (S) envelope glycoprotein, a protein sequence alignment was performed to compare the sequence of the Wuhan-Hu-1 strain of the novel coronavirus with that of the closely related human SARS-CoV S strain Tor2 sequence (Supplementary Figure 1).

The overall percent protein sequence identity found by the alignment was 76% (Figure 1(a)). A breakdown of the functional domains of the S protein, based on the SARS-CoV S sequence, reveals that the S1 receptor-binding domain was less conserved (64% identity) than the S2 fusion domain (90% identity).

Within S1, the NTD was found to be less conserved (51% identity) compared to the receptor binding domain (RBD; 74% identity), which is part of the C-terminal subdomain (Figure 1(a)).

The relatively high degree of sequence identity for the RBD is consistent with the view that SARS-CoV-2, like SARS-CoV, may use ACE2 as its host cell receptor [9,25,31]. Interestingly, when the more defined receptor binding motif (RBM) was analyzed (i.e. the region of SARS-CoV S containing residues that were shown to directly contact the ACE2 receptor) the identity between the two sequences drops to 50% (Figure 1(a)), in this case hinting at possible differences in binding residues involved in the interaction with the receptor and/or binding affinities [[31], [32], [33]].

As expected, within the well-conserved S2 domain, subdomain identities were high for the fusion peptide region (FP, 93% identity), high for the heptad-repeat 1 region (HR1, 88% identity), identical for HR2 (100% identity) and high for both the transmembrane and the C-terminal endodomain (TM, 93% identity; E, 97% identity) (Figure 1(a)).

Figure 1. Comparative analyses of SARS-CoV-2 S protein sequence.

(a) Protein sequence identities between SARS-CoV-2 S with SARS-CoV S. The S protein sequences were aligned using MAFFT and the sequence identities obtained for the full-length and domains/subdomains are shown on the S protein diagram. Amino acid numbering and delineations of domains and subdomains are based on the SARS-CoV S protein.

(b) Phylogenetic analysis of SARS-CoV-2 S protein. The S protein sequence of 15 isolates of SARS-CoV-2 was aligned using MAFFT with representatives of all four Betacoronavirus lineages. A Maximum-Likelihood tree was generated based on the alignment. The tree was rooted using the alphacoronavirus HCoV-229E S sequence. Highly pathogenic betacoronaviruses SARS-CoV and MERS-CoV are highlighted (bold font) along with bat SARS-like coronaviruses closely related to SARS-CoV-2 (Bat-SL-CoVZC45, Bat-SL-CoVZXC21, and Bat-SL-RaTG13). Number at nodes indicates bootstrap support (100 replicates), and the scale bar indicates the estimated number of substitutions per site. Accession numbers of sequences used in the analyses are found in the Materials and Methods section.

Discussion

The current COVID-19 pandemic caused by SARS-CoV-2 is evidence of the potential of coronaviruses to continuously evolve in wild reservoirs and jump to new species. Our study aims to contribute to our understanding of the SARS-CoV-2 from a phylogenetic and structural point of view, focusing on the functional and the proteolytically sensitive sites of the S protein.

Using phylogenetic analysis, we showed that the SARS-CoV-2 S protein is closely related to other SARS-like viruses originating in bats (Figure 1(b)), which agrees with early similar reports [34]. The identity of the S protein of BatCoV-RaTG13 strain of bat coronavirus was shown to be 96.65%, suggesting this virus as the closest relative to SARS-CoV-2.

While the origin of the novel coronavirus appears to be in bat reservoirs, there is still no definitive evidence of the possible intermediate host that could transmit the virus to humans. Recent reports have suggested the Malayan pangolins as an intermediate host for the SARS-CoV-2.

In our analysis, we found that the pangolin spike sequences grouped in a subclade branching from RaTG13. We additionally observed that the 2019 (Guangdong) pangolin sequence appeared to branch off early in a distinct subclade from RaTG13.

Based on these findings, we hypothesize that despite of having a common origin in bats, the phylogenetic relationship between pangolin CoVs and SARS-CoV-2 is not sufficient to support the claim that pangolins harbor the direct precursor to the currently circulating human SARS-CoV-2. In fact, our analysis suggests that both humans and pangolins could be considered final hosts of their respective coronavirus.

In this study, we show the presence of a distinct insert that maps to the S1/S2 priming loop of the SARS-CoV-2 spike protein and is not shared with SARS-CoV or any SARS-related viruses in Betacoronavirus lineage B.

During the preparation of this manuscript, the cryo-electron microscopy structures of SARS-CoV-2 S have recently been determined [33,52,53]. These studies have revealed in detail the structure of the SARS-CoV-2 spike RBD and how it contacts the ACE2 host cell receptor with notable differences compared to SARS-CoV [54]. It has been described that the SARS-CoV RBM “down” conformation packs more closely to the NTD of the S protein [33].

In the same report, the SARS-CoV-2 RBD was shown to be angled to the center of the trimmer in its down conformation, which differs to the SARS-CoV RBM structure. Interestingly, we observed in our models that the RBD is predicted to pack similarly to SARS-CoV and the RBM is also predicted to organize as a flexible loop with similar structure despite the lower amino acid identity between these two proteins (Figure 6).

In a more recent report, it has been shown that ACE2-binding mode of both SARS-CoV and SARS-CoV-2 RBDs is nearly identical, which supports the claim that the flexibility in the RBM is key to compensate the amino acid differences between the two CoVs proteins and agrees with our predicted models [54].

One of the common difficulties of the CoVs S protein cryo-EM studies is the difficulty to solve proteolytically sensitive regions in the protein [39,55]. Since the S1/S2 region is proteolytically sensitive, it is common to introduce mutations in this site to prevent proteolytic priming and to allow efficient heterologous expression and purification [42].

This has resulted in a difficulty to solve the S1/S2 region in most of the available CoV S protein structures, with a few exceptions for the S proteins of viruses belonging to the Alphacoronavirus and Gammacoronavirus genera [40,44].

Considering that this region is proteolytically sensitive and has been shown to play a major role in the S protein function in other CoVs, the use of in silico modeling tools has become a useful alternative to study this region in the context of the structural organization of the protein [37,38].

The SARS-CoV-2 S protein is not an exception to this issue and the recently reported structures do not allow resolution of the S1/S2 priming loop and/or have the loop mutated. We used the structural modeling approach to better understand the organization of this region, which is not only suggested to be functionally active, but has been reported as one of the major differences between the SARS-CoV-2 and its closest relative RaTG13 [25,33].

We observed that the SARS-CoV-2 S1/S2 site is predicted to be organized as an exposed flexible loop, suggesting that the site is easily available for protease cleavage and suggesting a major role in SARS-CoV-2 S function (Figure 7).

The significance of the SARS-CoV-2 spike protein S1/S2 priming loop is yet to be explored experimentally, but we consider it may fundamentally change the entry pathway of this virus compared to other known viruses in Betacoronavirus lineage B.

The presence of the extended S1/S2 priming loop containing paired basic residues predicts that SARS-CoV-2 S would most likely be cleaved by Golgi-resident proprotein convertases such as furin during virus assembly and delivery to the cell surface.

Indeed, analysis of Western blots of VSV-pseudoparticles containing SARS-CoV-2 S have shown the presence of cleaved S, in contrast to pseudoparticles containing SARS-CoV S [25].

In the case of MERS-CoV, but not SARS-CoV, it is known that priming of S by “pre-cleavage” occurs at the S1/S2 site, giving SARS-CoV-2 cleavage activation properties more in line with MERS-CoV than SARS-CoV [25,[56], [57], [58]].

The extended structural loop may also allow enhanced priming by trypsin-like proteases (TTSPs) or even cathepsins. SARS-CoV-2 is currently believed to be highly SARS-CoV-like with respect to its receptor binding, and the modeling studies reported here are broadly in line with this finding despite the relatively low amino acid identity in the RBM.

However, it is important to remember that changes in protease usage may allow coronaviruses to undergo receptor-independent entry (virus–cell fusion) as well as affect syncytia formation (cell–cell fusion) and tissue pathology [[59], [60], [61]].

Our study provides a structural context to the S1/S2 insert, which has also been reported by others [52,62]. The presence of a distinct insert containing paired basic residues in the S1/S2 priming loop is common in many coronaviruses in Betacoronavirus lineage C (e.g. MERS-CoV), as well as in lineage A (e.g. mouse hepatitis virus, MHV) and lineage D, and is universally found in Gammacoronavirus S (e.g. IBV) [40].

It is noticeably absent in most Alphacoronaviruses, with the clear exception of type I canine and feline coronaviruses [28,37]. One feature of the distinct insert for of SARS-CoV-2 that warrants attention relates to potential changes as the virus evolves.

An equivalent loop is present in influenza HA (in this case adjacent to the fusion peptide), and insertions of basic residues into the loop are a primary marker of conversion from low pathogenicity to highly pathogenic avian influenza virus (e.g. H5N1) [63].

In coronaviruses, such loop modifications are known to affect MHV pathogenesis and to modulate neurovirulence and neuroinvasiveness of HCoV-OC43 [64,65]. The FCoV is another example where S1/S2 loop modifications appear to lead directly to changes in viral pathogenesis [37,66,67].

In the case of FCoV, the equivalent proteolytically sensitive structural loop is within a hypervariable region of the spike gene, suggesting that this region of spike is a significant driver of virus evolution [67].

At present, SARS-CoV-2 is behaving in a distinct manner compared to SARS-CoV. We believe our findings are of special importance considering that the available data indicates ACE2 as a suitable cellular receptor for SARS-CoV-2 entry [48,68].

In our modeling analysis, we observed that the RBM of the SARS-CoV-2 predicted a similar organization as SARS-CoV and that deletions at this RBM region in other bat-CoVs are reported to not impact its ability to bind ACE2 [[49], [50], [51]].

This suggests that instead of receptor binding, the S1/S2 loop is a distinctive feature relevant to SARS-CoV-2 pathogenesis and marks a unique similarity to MERS-CoV. We would predict that the distinct insert in SARS-CoV-2 S would give the virus biological properties more in line with MERS-CoV and not SARS-CoV, especially with regard to its cell entry pathway. However, it may also impact virus spread and transmission.

While many epidemiological features of SARS-CoV-2 still need to be resolved, there are many features of transmission that appear to align more with MERS-CoV than SARS-CoV.

One component of transmission is the reproductive number (R0), which is currently thought to be approximately 2.0–3.0 for SARS-CoV-2, broadly in line with than for SARS-CoV, and while MERS-CoV has a low R0 in humans (< 1), it is high in camels and in outbreak situations (> 3) [[69], [70], [71], [72], [73]].

Another study has reported that the serial interval (time from the disease onset in a patient to the onset of the disease in secondary case) can be estimated to be below 4 days, suggesting that transmission can occur before the onset of clinical signs [74]. These two parameters highlight the high transmissibility of the SARS-CoV-2. One notable feature of the S protein S1/S2 cleavage site was first observed during the purification of the MHV S protein for structural analysis [42].

MHV with an intact cleavage loop was unstable when expressed, and so we consider that the S1/S2 loop controls virus stability, likely via access to the down-stream S2′ site that regulates fusion peptide exposure and activity. As such, it will interesting to monitor the effects of S1/S2 loop insertions and proteolytic cleavability in the context of virus transmission, in addition to virus entry, pathogenesis, and evolution.

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Cornell University